Genetic insulator for preventing influence by another gene promoter

ABSTRACT

A 16 bp polynucleotide sequence of  Arabidopsis thaliana  is a genetic insulator that can effectively isolate a transgene from positional effects of neighboring gene activities in transgenic plant cells.

CLAIM OF PRIORITY

[0001] This application claims priority to U.S. provisional patentapplication Ser. No. 60/241,735, filed Oct. 20, 2000.

FIELD OF THE INVENTION

[0002] The invention relates to the field of plant molecular biology andbiotechnology. Specifically, the invention relates to polynucleotidesequences that can minimize or eliminate the position effect on atransgene in a plant.

BACKGROUND OF THE INVENTION

[0003] Transgenic technology is widely used in biotechnology. Ineukaryotes, the temporal and spatial expression of transgenes isregulated by transcription factors through their interaction withenhancer elements. However, eukaryotic enhancer-promoter interactionslack the specificity for precise temporal and spatial patterns oftransgenic expression. The expression of a transgene can be affected by(a) the promoter of a selectable marker gene that is closely linked withthe transgene, and/or (b) by chromosomal genes that flank the transgene,which is often referred to as the “position effect”. These effects areoften not desirable, especially when tissue-specific, preciselycontrolled, or optimal transgene expression is wanted.

[0004] It is known that a eukaryotic genome has organizationalproperties that rely on the ability of the chromosome to establishautonomous functional units. The polynucleotide sequences that separatethese domains are called genetic insulator elements. These geneticinsulator elements can buffer a transgene from position effects, so thatan introduced transgene can be expressed independent of its location inthe chromosome. In addition, a genetic insulator may repress nonspecificinteractions between enhancers and promoters. Thus, it could be possibleto obtain precise gene expression by using an appropriate geneticinsulator to shield the effects of neighboring gene promoters.

[0005] Genetic insulators in fruit fly (Drosophila) include specializedchromatin structures (scs and scs′), which consist of 350 base pairs(bp) and 200 bp, respectively. These sequences are associated withchromatin structures and serve as boundaries that can prevent activationby enhancer elements. Similarly, genetic insulators are known in thechicken in the form of lysozyme “A” element and the β-globin LCR (HS4),which contain 242 bp. These insulators generally comprise 200-250 bp andfunction directionally. Additionally, the gypsy chromatin insulator ofDrosophila (originally isolated from the gypsy retrotransposon) protectsa gene and its regulatory elements from both positive and negativeposition effects (see, U.S. Pat. No. 6,229,070, incorporated byreference).

[0006] By contrast, there previously have been no genetic insulatorsisolated from plants. There is a need in the art to develop a plantgenetic insulator to control transgene expression in plants.

SUMMARY OF THE INVENTION

[0007] The invention provides an isolated polynucleotide that containsat least one copy of either a polynucleotide region having the sequenceset forth in SEQ ID NO:9 (5′GAATATATATATATTC3′) or a polynucleotideregion having a sequence that is a variant or fragment of the sequenceset forth in SEQ ID NO:9, wherein the polynucleotide region has agenetic insulator activity. In various embodiments, the polynucleotidehas a sequence as set forth in SEQ ID NOS:1, 5, 9, 10, 11, 12, 15, 16,17, 18, 21, 22, 23, 24, 25, 26, 27, 28, 30, 31, 33, 34, 35 or 36. In oneembodiment, the plant genetic insulator sequence contains only 16 basepair (bp), and is completely distinct in size and function from thegenetic insulators of fruit fly and chicken.

[0008] The invention provides a recombinant polynucleotide containing aplant genetic insulator comprising at least one copy of a polynucleotidehaving the sequence set forth in SEQ ID NO:9 (5′GAATATATATATATTC3′) or apolynucleotide region having a sequence that is a variant or fragment ofthe sequence set forth in SEQ ID NO:9, wherein the polynucleotide regionhas a genetic insulator activity.

[0009] The invention also provides a vector, comprising: a replicablevector; and the nucleic acid mentioned above that is inserted into thevector. Preferably, the vector is an expression vector, a plant vector,or a plant expression vector. The invention also provides a host cell,in which the vector is situated. The host cell may be a plant cell or amicroorganism.

[0010] The invention further provides a transgenic plant containing thegenetic insulator polynucleotide. The invention is also directed to arecombinant seed containing the genetic insulator polynucleotide.

[0011] The invention provides a method for expressing a polypeptide inan organism comprising: constructing a vector comprising the geneticinsulator polynucleotide; inserting the vector into the organism,wherein the genetic insulator sequence is recombined into the organism;and allowing the organism to express the polypeptide. In this method,the polypeptide may be encoded on an insert in the vector.Alternatively, the polypeptide may be encoded on the genome of organism.Further, the described nucleic acid may be inserted immediately upstreamof the nucleic acid encoding the polypeptide. Preferably, the organismis a plant. More preferably, the plant may be Arabidopsis or tobacco.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012]FIG. 1 is a schematic drawing showing constructs used toinvestigate the buffering role of the insulator. 1× or 2× represents 1copy or 2 copies, respectively. lacO, the mutated 18-bp lac Operatorsequence that possesses a perfect palindromic structure. GUS, thereporter gene encoding β-glucuronidase. NPT II, a gene encoding neomycinphosphotransferase II that renders transgenic plant cells resistant tokanamycin. Ω is the TMV Ω leader sequence. P_(35S) is the cauliflowermosaic virus (CaMV) 35S promoter. P_(35Smini-35)is the minimum promoter(−35 region) of the CaMV 35S promoter. RB and LB are the right and leftborder of T-DNA, respectively.

[0013]FIG. 2 is a set of photographs showing the expression of thereporter gene GUS in 2-week old transgenic Arabidopsis seedlingsharboring (A) pGL429, (B) pGL450, (C) pGL419, (D) pGL469, (E) pGL443, or(F) pGL446. The inserts are close-ups of respective transgenic seedlingsafter staining with the GUS substrate X-gluc. Note the 3 inserts showingdifferent staining patterns in Panel E (about 68% of the plants in PanelE were not stained).

[0014]FIG. 3 shows the formation of the insulator-nuclear proteincomplex (CX) revealed by electrophoretic mobility shift assay (EMSA).FIG. 3A is an autoradiograph of EMSA. 5 μg (Lane 2) or 9 μg (Lanes 3-7)of Arabidopsis nuclear proteins (NPs) were co-incubated with ³²P-labeledinsulator (NI29) or lacO (L31), respectively. There was 0- (Lane 3), 2-(Lane 4), 20- (Lane 5), or 200-fold (Lane 6) molar excess of cold NI29competitor. FIG. 3B is a bar graph showing the relative amount of CXformed in each lane. The amount of CX in Lane 3 was set as 100 FP arethe free probes. + or − indicate with or without NPs added,respectively.

[0015]FIG. 4 shows the effects of mutations in the insulator on CXformation. FIG. 4A is a set of the sequences of DNA probes. Only onestrand is shown. The capital letters are the perfect palindromicsequence or its derivatives/mutants. The small letters represent thenative sequences flanking the palindrome in Arabidopsis. NI29, thenative insulator. NIm, naturally occurring mutant of NI29. M1-M7 areartificial mutants/derivatives of NI29. FIG. 4B and FIG. 4C areautoradiographs of EMSA. FIG. 4D and FIG. 4E are bar graphs showing thequantitative analysis of CX shown in FIG. 4B and FIG. 4C, respectively.The amount of CX formed in each lane was normalized to the amount of CXformed in Lane 2 of respective autoradiographs. Note the scaledifference of the Y-axes between FIG. 4D and FIG. 4E. CX is theinsulator-nuclear protein complex. FP are the free probes. NPs are theArabidopsis nuclear proteins. + or − indicate with or without NPs added,respectively.

DETAILED DESCRIPTION OF THE INVENTION

[0016] The invention is directed to a polynucleotide cloned fromArabidopsis thaliana, and variants thereof, that possesses geneticinsulating activity. In one embodiment, the specifically exemplifiedinsulator sequence is a polynucleotide having a 16 bp sequence thatdisplays a perfect palindrome structure (5′GAATATATATATATTC3′; SEQ IDNO:9).

[0017] As used herein, the terms “insulator”, “genetic insulator” and“insulator sequence” refer to a nucleic acid sequence that, when it isinserted upstream of a gene of interest, prevents the influence of othernearby regulatory sequences in the expression of the gene of interest.The term “insulator” (or “genetic insulator”; “chromatin insulator”, or“boundary element”) thus has its conventional meaning in the art, andrefers to a DNA segment that prevents enhances located on one side ofthe insulator or boundary element from acting on promoters located inthe adjacent domain (see, U.S. Pat. No. 6,037,525, incorporated byreference).

[0018] Preferably, the genetic insulator is a plant insulator. The term“plant insulator” means that the insulator is functional in a plant, andincludes insulators isolated from plants. Plant genetic insulators ofthe invention may be taken from any suitable plant, including thoseplants specified below; but insulator with appropriate sequences may betaken from any suitable animal including insects (e.g., Drosophila),mammals (e.g., rat, mouse, dog, cat), birds (e.g., chicken, turkey),etc.; and insulators may be taken from other eukaryotes such as fungi(e.g., Saccharomyces cereviceae). Where two insulators are employed,they may be the same or different sequences. The insulator may be avariant of a naturally occurring insulator, so long as it retainsfunction as an insulator.

[0019] Sequence Identity. The exemplified insulator sequence(5′GAATATATATATATTC3′; SEQ ID NO:9) has been isolated from Arabidopsis.However, other variations and analogs that have the desired effect ofallowing the transgene of interest to be expressed from its specifiedcontrol elements without the influence of neighboring regulatoryelements are also within the purview of the invention (for some of theacceptable variants, see TABLE 3, below). This disclosure sets forthseveral examples of experiments in which the insulator sequences aremutated and their ability to insulate the gene of interest is assayed.Thus, the insulator may be any variant or fragment of the exemplifiedsequence that has insulator activity. Also, the insulator may be anyvariant or fragment of the exemplified sequence that hybridizes to thepolynucleotide exemplified sequence under 5×SSC and 42° C. washconditions (see, Sambrook et al., Molecular Cloning: A LaboratoryManual, 2d Ed. (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,1989)).

[0020] Two polynucleotide sequences are said to be “identical” if thesequence of residues is the same when aligned for maximum correspondenceas described below. The term “complementary” applies to nucleic acidsequences and is used herein to mean that the sequence is complementaryto all or a portion of a reference polynucleotide sequence.

[0021] Optimal alignment of sequences for comparison can be conducted bythe local homology algorithm of Smith & Waterman, Add. Appl. Math.,2:482 (1981), by the homology alignment method of Needleman & Wunsch, J.Mol. Biol., 48:443 (1970), by the search for similarity method ofPearson & Lippman, Proc. Natl. Acad. Sci. USA, 85:2444 (1988), or thelike. Computer implementations of the above algorithms are known as partof the Genetics Computer Group (GCG) Wisconsin Genetics Software Package(GAP, BESTFIT, BLASTA, FASTA and TFASTA), 575 Science Drive, Madison,Wis.

[0022] “Percentage of sequence identity” is determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the sequence in the comparison window may comprise additionsor deletions (i.e. “gaps”) as compared to the reference sequence foroptimal alignment of the two sequences being compared. The percentageidentity is calculated by determining the number of positions at whichthe identical residue occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the window and multiplying the result by 100 toyield the percentage of sequence identity. Total identity is thendetermined as the average identity over all of the windows that coverthe complete query sequence.

[0023] Use of the Genetic Insulator of the Invention. One of the mainproblems of current transgenic techniques is the gradual loss ofexpression of the transfected gene, perhaps due to the repressiveinfluence of the DNA sequences that surround the integration site of thetransfected gene. By insulating a gene to be transfected with thegenetic insulator of the invention, the gene could be usefullymaintained in an active state.

[0024] The insulator sequence can be placed upstream or downstream ofthe gene of interest to have the insulating effect on the gene. Forexample, the insulator sequence may be positioned upstream of the geneof interest. At least one copy of the insulator sequence may be used inthe invention.

[0025] While the invention can be used to specifically to tightlycontrol the expression of transgenes, it is also possible to use theinsulator sequence to more tightly control endogenous genes by insertingthe insulator sequence upstream of the endogenous gene of interest.

[0026] Advantageously, the function of the insulator element isindependent of its orientation, and thus the insulator can function whenplaced in genomic or reverse genomic orientation with respect to thetranscription unit. Also, only one copy of the insular need beintroduced into the plant to achieve effective insulator function(contrast, U.S. Pat. No. 6,037,525, incorporated by reference.)

[0027] The genetic insulator of the invention can also be a useful toolin gene regulation studies and in the production of stably transfectedcell lines. Because the expression of a stably transfected gene isinfluenced by adjacent regulatory elements near the site of geneintegration, insulating the transfected gene with the genetic insulatorof the invention eliminates the variability that is caused bycell-to-cell differences in integration position and in the random sitesof integration. This should obviate the need for numerous founder linesof clonal cell lines.

[0028] Polynucleotide Constructs. A variety of enhancers, promoters, andgenes are suitable for use in the constructs of the invention, and thatthe constructs will contain the necessary start, termination, andcontrol sequences for proper transcription and processing of the gene ofinterest when the construct is introduced into a mammalian or a highereukaryotic cell. DNA constructs known as “expression cassettes,”preferably include a transcription initiation region, a structural gene(a structural polynucleotide coding for a polypeptide) positioneddownstream from the transcription initiation region and operativelyassociated therewith, an insulator positioned: (i) 5′ to thetranscription initiation region, (ii) 3′ to the structural gene, or(iii) both 5′ to the transcription initiation region and 3′ to thestructural polynucleotide coding for a polypeptide, and, optionally, atermination sequence including stop signal for RNA polymerase and apolyadenylation signal for polyadenylase. The promoter should be capableof operating in the cells to be transformed. The termination region maybe derived from the same gene as the promoter region, or may be derivedfrom a different gene.

[0029] The term “operatively associated,” as used herein, refers topolynucleotide regions on a single polynucleotide molecule that areassociated, so that the function of one affects the finction of theother. Thus, a transcription initiation region is operatively associatedwith a structural gene when it is capable of affecting the expression ofthat structural gene (i.e., the structural gene is under thetranscriptional control of the transcription initiation region). Inother words, the polynucleotide sequences described herein are “operablylinked” with other polynucleotide sequences. DNA regions are operablylinked when they are functionally related to each other. For example,DNA for a presequence or secretory leader is operably linked to DNA fora polypeptide if it is expressed as a preprotein which participates inthe secretion of the polypeptide; a promoter is operably linked to acoding sequence if it controls the transcription of the sequence; or aribosome binding site is operably linked to a coding sequence if it ispositioned so as to permit translation. Generally, operably linked meanscontiguous (or in close proximity to) and, in the case of secretoryleaders, contiguous and in reading phase. The transcription initiationregion is said to be “upstream” from the structural gene, which is inturn said to be “downstream” from the transcription initiation region.

[0030] The transcription initiation region, which includes the RNApolymerase binding site (promoter), may be native to the host plant tobe transformed or may be derived from an alternative source, where theregion is functional in the host plant. Other sources include theAgrobacterium T-DNA genes, such as the transcriptional initiationregions for the biosynthesis of nopaline, octapine, mannopine, or otheropine transcriptional initiation regions; transcriptional initiationregions from plants, such as the ubiquitin promoter; root specificpromoters; transcriptional initiation regions from viruses (includinghost specific viruses), or partially or wholly synthetic transcriptioninitiation regions. Transcriptional initiation and termination regionsare well known. The transcriptional initiation regions may, in additionto the RNA polymerase binding site, include regions that regulatetranscription, where the regulation involves, for example, chemical orphysical repression or induction. Thus, the transcriptional initiationregion, or the regulatory portion of such region, is obtained from anappropriate gene that is so regulated.

[0031] The term “structural gene” refers to those portions of geneswhich comprise a DNA segment coding for a protein, polypeptide, orportion thereof, possibly including a ribosome binding site and/or atranslational start codon, but lacking a transcription initiationregion. The term can also refer to transgenic copies of a structuralgene not naturally found within a cell but artificially introduced. Thestructural gene may encode a protein not normally found in the plantcell in which the gene is introduced or in combination with thetranscription initiation region to which it is operationally associated,in which case it is termed a heterologous structural gene. Genes thatmay be operationally associated with a transcription initiation regionof the present invention for expression in a plant species may bederived from a chromosomal gene, cDNA, a synthetic gene, or combinationsthereof.

[0032] The term “transgene” refers to a gene that is artificiallytransferred into, and maintained, and may be expressed in host organismssuch as plants. Various techniques are amply exemplified in theliterature and find particular exemplification in Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2d Ed. (Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., 1989) and other standard texts.

[0033] As used herein, a transgenic plant refers to a plant in which atleast some cells are stably transformed with a heterologous DNAconstruct. As used herein, a heterologous DNA construct refers to DNAthat is artificially introduced into a cell or into a cell's ancestor.Such DNA may contain genes or DNA which would not normally be found inthe cell to be transformed, or may contain genes or DNA which iscontained in the cell to be transformed. In the latter case, cells aretransformed so that they contain additional or multiple copies of theDNA sequence or gene of interest. DNA constructs may be introduced intocells by a variety of gene transfer methods known to those skilled inthe art, for example, gene transfection, microinjection,electroporation, and infection.

[0034] The polynucleotide constructs of the invention may be used ingene transfer methods to allow the protected expression of one or moregiven genes that are stably transfected into the cellular plant DNA(“recombinant” polynucleotides of the invention). Recombinantpolynucleotide constructs comprising one or more of the geneticinsulator polynucleotide sequences described herein and an additionalpolynucleotide sequence are included within the scope of this invention.These recombinant DNA constructs have sequences that (1) do not occur innature; (2) exist in a form that does not occur in nature; or (3) existin association with other materials that do not occur in nature. Theconstructs of the invention would not only insulate a transfected geneor genes from the influences of DNA surrounding the site of integration,but would also prevent the integrated constructs from impacting on theDNA at the site of integration and would therefore prevent activation ofthe transcription of genes that are harmful or detrimental to the cell.

[0035] Transcriptionally competent transcription units can be made byconventional techniques (see, Sambrook et al., Molecular Cloning: ALaboratory Manual, 2d Ed. (Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 1989)). The comparatively small size of the insulator ofthe invention (contrast, U.S. Pat. Nos. 5,610,053 and 6,037,525, eachincorporated by reference) makes for easier use of the insulatorsequences. In general, the insulator of the invention is placed insufficient proximity to the enhancer so that it is functionally activeto buffer the effects of a cis-acting DNA region on the promoter of thetranscription unit. However, in some cases, the insulator can be placeddistantly from the transcription unit. The optimal location of theinsulator element can be determined by routine experimentation for anyparticular DNA construct, with additional guidance provided by thelocation of the genetic insulator in non-transgenic plant DNA, forexample, in Arabidopsis DNA.

[0036] Vectors. The invention is further directed to a replicable vectorcontaining the insulator sequence and cDNA which may code for apolypeptide and which is capable of expressing the polypeptide under thetranscriptional control of a promoter. The vector is transferable to thehost organism. Preferably, the host organism is a plant or plant cell.The vector may be an integrating or non-integrating vector and isconveniently a plasmid.

[0037] Vectors that may be used to transform plant tissue with DNAconstructs of the invention include Agrobacterium vectors,non-Agrobacterium vectors, as well as other known plant vectors suitablefor DNA-mediated transformation. In general, Agrobacterium vectorscomprise an agrobacterium, typically Agrobacterium tumefaciens, thatcarry at least one tumor-inducing (or “Ti”) plasmid. When theagrobacterium is Agrobacterium rhizogenes, this plasmid is also known asthe root-inducing (or “Ri”) plasmid. The Ti (or Ri) plasmid contains DNAreferred to as “T-DNA” that is transferred to the cells of a host plantwhen that plant is infected by the agrobacterium. In an Agrobacteriumvector, the T-DNA is modified by genetic engineering techniques tocontain the “expression cassette”, or the gene or genes of interest tobe expressed in the transformed plant cells, along with the associatedregulatory sequences. Such Agrobacterium vectors are useful forintroducing foreign genes into a variety of plant species, and areparticularly useful for the transformation of dicots.

[0038] Vector Containing a Particular Genetic Insulator Sequence. Toinvestigate the function of the 16 bp sequence (SEQ ID NO:9), we firstmade a construct called pGL419 (construct 3 in FIG. 1) in which twocopies of the 16 bp palindrome were fused with a 35S minimumpromoter-GUS. As controls, we constructed pGL429 (construct 1 in FIG. 1;a bidirectional promoter construct shown in a separate disclosure) andpGL450 (which is essentially the same as pGL419 except that 2 copies ofmutated 18 bp lac operator sequence were used instead of the 16 bpinsulator sequence). Like the 16 bp insulator, the mutated lac operatoris a perfect palindrome.

[0039] Transgenic Arabidopsis plants containing either pGL429 or pGL450showed constitutive GUS expression. However, none of the transgenicplants harboring pGL419 showed any GUS expression, suggesting that the16 bp insulator can insulate the GUS gene from the enhancers of the 35Spromoter. Moreover, by constructing pGL469 (construct 4 in FIG. 1), wefound that one copy of the 16 bp insulator is sufficient for theinsulation activity.

[0040] Additional details of construction of vectors containing aninsulator of the invention (including pGL419 and pGL469) is provided inEXAMPLE 1.

[0041] Methods for the Demonstration of Insulator Function. To test acandidate insulator for activity as an insulator, one may simply clonethe candidate insulator into a construct comprising, 5′ to 3′, aconstitutive enhancer, the candidate insulator, an inducible promoter(e.g., an HSP70 promoter), and a reporter gene (e.g., GUS orluciferase). Plant cells are then transformed with the construct by anysuitable means as described herein, and (optionally) plants created fromthe cells. See, EXAMPLE 1, below.

[0042] More particularly, a position effect on a transgene is generallyexerted by the promoters/enhancers of neighboring genes. This positioneffect can be demonstrated in either of the following two ways.

[0043] One way is to use a constitutive promoter fused to the reportergene GUS. Transgenic plants containing this construct should display aspectrum of the GUS expression level, with some plants expressing GUS ata very high level and some other plants at lesser levels. The additionof the insulator sequence at the 5′ end of the constitutive promotershould result in an even level of GUS expression with no or littlevariation among all transgenic plants.

[0044] An alternate way to demonstrate the position effect is asfollows. We made a construct called pGL443 (construct 5 in FIG. 1) wherewe used the 35S minimum promoter-GUS gene. The 35S minimum promoteritself has no transcription activity. When this construct is randomlyinserted into a plant genome, some neighboring gene promoters/enhancersin certain transgenic plants may activate the GUS expression dependingon the activities of the neighboring gene promoters. As shown in FIG. 2,32% of transgenic Arabidopsis seedlings (2 weeks old) containing pGL443showed the GUS expression exhibiting the position effect. Note that noGUS expression would have been observed if there were no positioneffect, because the minimum promoter itself has no transcriptionactivity. However, when two copies of the insulator sequence are addedat the 5′ end of the minimum promoter-GUS gene (pGL446), the transgenicplants did not show GUS expression, demonstrating that the insulatorsequence can serve as a buffer to eliminate or reduce the positioneffect.

[0045] Transformed Cells. The invention further relates to a transformedcell or microorganism containing cDNA or a vector which codes for thepolypeptide or a fragment or variant thereof and which is capable ofexpressing the polypeptide. In one embodiment, the transformed cell is aseed cell.

[0046] Plant Cell Expression Systems. Transgenic plants may be producedusing the DNA constructs of the invention by the DNA-mediatedtransformation of plant cell protoplasts and subsequent regeneration ofthe plant from the transformed protoplasts in accordance with procedureswell known in the art. Any plant tissue capable of subsequent clonalpropagation, whether by organogenesis or embryogenesis, may betransformed with a vector of the present invention. The term“organogenesis,” as used herein, means a process by which shoots androots are developed sequentially from meristematic centers; the term“embryogenesis,” as used herein, means a process by which shoots androots develop together in a concerted fashion (not sequentially),whether from somatic cells or gametes. The particular tissue chosen willvary depending on the clonal propagation systems available for, and bestsuited to, the particular species being transformed. Exemplary tissuetargets include leaf disks, pollen, embryos, cotyledons, hypocotyls,megagametophytes, callus tissue, existing meristematic tissue (e.g.,apical meristems, axillary buds, and root meristems), and inducedmeristem tissue (e.g., cotyledon meristem and hypocotyl meristem).

[0047] Plants of the invention may take a variety of forms. The plantsmay be chimeras of transformed cells and non-transformed cells; theplants may be clonal transformants (e.g., all cells transformed tocontain the expression cassette); the plants may comprise grafts oftransformed and untransformed tissues (e.g., a transformed root stockgrafted to an untransformed scion in citrus species). The transformedplants may be propagated by a variety of means, such as by clonalpropagation or classical breeding techniques.

[0048] In plants, transformation vectors capable of introducingpolynucleotides containing the insulator sequence are easily designed,and generally contain one or more DNA coding sequences of interest underthe transcriptional control of 5′ and 3′ regulatory sequences. Suchvectors generally comprise, operatively linked in sequence in the 5′ to3′ direction, an insulator sequence and a promoter sequence that directsthe transcription of a downstream heterologous structural DNA in aplant; optionally a 5′ non-translated leader sequence; a nucleotidesequence that encodes a protein of interest; and a 3′ non-translatedregion that encodes a polyadenylation signal which functions in plantcells to cause the termination of transcription and the addition ofpolyadenylate nucleotides to the 3′ end of the MRNA encoding saidprotein. Plant transformation vectors also generally contain aselectable marker. Typical 5′-3′ regulatory sequences include atranscription initiation start site, a ribosome binding site, an RNAprocessing signal, a transcription tennination site, and/or apolyadenylation signal. Vectors for plant transformation are described(Schardl et al., Gene 61, 1-14, (1987); Plant Mol Biol., 25:989-994(1994)). Particularly useful vectors for this invention include, but arenot limited to pPZP family.

[0049] Plant Transformation and Regeneration. A variety of differentmethods can be employed to introduce such vectors into plant trichome,protoplasts, cells, callus tissue, leaf discs, meristems, etc., togenerate transgenic plants, including Agrobacterium-mediatedtransformation, particle gun delivery, microinjection, electroporation,polyethylene glycol-mediated protoplast transformation,liposome-mediated transformation, etc. In general, transgenic plantscomprising cells containing and expressing polynucleotides encodingvarious enzymes can be produced by transforming plant cells with a DNAconstruct as described above via any of the foregoing methods; selectingplant cells that have been transformed on a selective medium;regenerating plant cells that have been transformed to producedifferentiated plants; and selecting a transformed plant which expressesthe enzyme-encoding nucleotide sequence.

[0050] The polynucleotides can be introduced either in a singletransformation event (all necessary polynucleotides present on the samevector), a co-transformation event (all necessary polynucleotidespresent on separate vectors that are introduced into plants or plantcells simultaneously), or by independent transformation events (allnecessary polynucleotides present on separate vectors that areintroduced into plants or plant cells independently). Traditionalbreeding methods can subsequently be used to incorporate the entirepathway into a single plant. Specific methods for transforming a widevariety of dicots and obtaining transgenic plants are well documented inthe literature.

[0051] Successful transformation and plant regeneration of transgenicplants have been achieved in the monocots as follows: asparagus(Asparagus officinalis; Bytebier et al, Proc. Natl. Acad. Sci. USA,84:5345 (1987)); barley (Hordeum vulgarae; Wan & Lemaux, Plant Physiol.,104:37 (1994)); maize (Zea mays; Rhodes et al, Science, 240:204 (1988);Gordon-Kamm et al, Plant Cell, 2:603 (1990); Fromm et al,Bio/Technology, 8:833 (1990); Koziel et al, Bio/Technology, 11:194(1993)); oats (Avena saliva; Somers et al, Bio/Technology, 10:1589(1992)); orchardgrass (Dactylic glomerata; Horn et al, Plant Cell Rep.,7:469 (1988)); rice (Oryza saliva, including indica and japonicavarieties; Toriyama et al, Bio/Technology, 6:10 (1988); Zhang et al,Plant Cell Rep., 7:379 (1988); Luo & Wu, Plant Mol. Biol. Rep., 6:165(1988); Zhang & Wu, Theor. Appl. Genet., 76:835 (1988); Christou et al,Bio/Technology, 9:957 (1991)); rye (Secale cereale; De la Pena et al,Nature, 325:274 (1987)); sorghum (Sorghum bicolor; Cassas et al, Proc.Natl. Acad. Sci. USA; 90:11212 (1993)); sugar cane (Saccharum spp.;Bower & Birch, Plant J., 2:409 (1992)); tall fescue (Festucaarundinacea; Wang et al, Bio/Technology, 10:691 (1992)); turfgrass(Agrostis palustris; Zhong et al, Plant Cell Rep., 13:1 (1993)); andwheat (Triticum aestinum; Vasil et al, Bio/Technology, 10:667 (1992);Weeks et al, Plant Physiol., 102:1077 (1993); Becker et al, Plant J.,5:299 (1994)).

[0052] Relevant Plants. The methods of the invention can be carried outwith cells from a variety of different plants. As used herein, the term“plant” or “plants” means vascular plants, including both monocots anddicots, and both angiosperms and gymnosperms. Particularly useful plantsfor exploiting the genetic insulator sequences of the invention includeplant and ferns of the genus Populus, Ermophilia, Lycopersicon,Nicotiana, Cannabis, Pharbitis, Apteria, Psychotria, Mercurialis,Chrysanthemum, Polypodium, Pelargonium, Polytrichiales, Mimulus,Chamomile, Monarda, Solanum, Achillea, Valeriana, Ocimum, Medicago,Aesculus, Newcastelia, Plumbago, Pityrogramma, Phacelia, Avicennia,Tamarix, Frankenia, Limonium, Foeniculum, Thymus, Salvia, Kadsura,Beyeria, Humulus, Mentha, Artemisia, Nepta, Geraea, Pogogstemon,Majorana, Cleome, Cnicus, Parthenium, Ricinocarpos, Parthenium,Hymenaea, Larrea, Primula, Phacelia, Dryopteris, Plectranthus,Cypripedium, Petunia, Datura, Mucuna, Ricinus, Hypericum, Myoporum,Acacia, Diplopeltis, Dodonaea, Halgania, Cyanostegia, Prostanthera,Anthocercis, Olearia, and Viscaria. Plants which may be employed inpracticing the present invention include (but are not limited to)tobacco (Nicotiana tabacum), potato (Solanum tuberosum), soybean(Glycine max), peanuts (Arachis hypogaea), cotton (Gossypium hirsutum),sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee(Cofea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus),citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camelliasinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficuscasica), guava (Psidium guajava), mango (Mangifera indica), olive (Oleaeuropaea), papaya (Carica papaya), cashew (Anacardium occidentale),macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugarbeets (Beta vulgaris), corn (Zea mays, also known as maize), wheat,oats, rye, barley, rice, vegetables, ornamentals, and conifers.Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g.,Lactuea sativa), green beans (Phaseolus vulgaris), lima beans (Phaseoluslimensis), peas (Pisum spp.) and members of the genus Cucumis such ascucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon (C.melo). Ornamentals include azalea (Rhododendron spp.), hydrangea(Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosaspp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias(Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia(Euphorbia pulcherima), and chrysanthemum. Gymnosperms which may beemployed to carrying out the present invention include conifers,including pines such as loblolly pine (Pinus taeda), slash pine (Pinuselliotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinuscontorta), and Monterey pine (Pinus radiata); Douglas-fir (Pseudotsugamenziesii); Western hemlock (Tsuga canadensis); Sitka spruce (Piceaglauca); redwood (Sequoia sempervirens); true firs such as silver fir(Abies amabilis) and balsam fir (Abies balsamea); and cedars such asWestern red cedar (Thuja plicata) and Alaska yellow-cedar (Chamaecyparisnootkatensis).

[0053] Production of Transgenic Plants Comprising a Gene or MultipleGenes ofInterest. Plant transformation vectors capable of deliveringpolynucleotides (genomic DNAs, plasmid DNAs, cDNAs, or synthetic DNAs)can be easily designed. Various strategies can be employed to introducethese DNAs to produce transgenic plants capable of biosynthesizing highlevels of a gene product of interest including:

[0054] 1. Transforming individual plants with an encoding DNA ofinterest. Two or more transgenic plants, each containing one of theseDNAs, can then be grown and cross-pollinated so as to produce hybridplants containing the two DNAs. The hybrid can then be crossed with theremaining transgenic plants in order to obtain a hybrid plant containingall DNAs of interest within its genome.

[0055] 2. Sequentially transforming plants with plasmids containing eachof the encoding DNAs of interest, respectively.

[0056] 3. Simultaneously cotransforming plants with plasmids containingeach of the encoding DNAs, respectively.

[0057] 4. Transforming plants with a single plasmid containing two ormore encoding DNAs of interest.

[0058] 5. Transforming plants by a combination of any of the foregoingtechniques in order to obtain a plant that expresses a desiredcombination of encoding DNAs of interest.

[0059] Traditional breeding of transformed plants produced according toany one of the foregoing methods by successive rounds of crossing canthen be carried out to incorporate all the desired encoding DNAs in asingle homozygous plant line (see, published PCT patent application WO93/02187).

[0060] The use of vectors containing different selectable marker genesto facilitate selection of plants containing two or moredifferent-encoding DNAs is advantageous. Examples of useful selectablemarker genes include those conferring resistance to kanamycin,hygromycin, sulphonamides, glyphosate, bialaphos, and phosphinothricin.

[0061] Stability for Transgene Expression. As several overexpressedenzymes may be required to produce optimal levels, the phenomenon ofco-suppression may influence transgene expression in transformed plants.Several strategies can be employed to avoid this potential problem.

[0062] One commonly employed approach is to select and/or screen fortransgenic plants that contain a single intact copy of the transgene orother encoding DNA. Agrobacterium-mediated transformation technologiesare preferred in this regard.

[0063] The use of enhancers from tissue-specific ordevelopmentally-regulated genes may ensure that expression of a linkedtransgene or other encoding DNA occurs in the appropriately regulatedmanner.

[0064] The use of different combinations of promoters, plastid targetingsequences, and selectable markers in addition to the trichome-specificregulatory sequence, for introduced transgenes or other encoding DNAscan avoid potential problems due to trans-inactivation in cases wherepyramiding of different transgenes within a single plant is desired.

[0065] Finally, inactivation by co-suppression can be avoided byscreening a number of independent transgenic plants to identify thosethat consistently overexpress particular introduced encoding DNAs.Site-specific recombination in which the endogenous copy of a gene isreplaced by the same gene, but with altered expression characteristics,should obviate this problem.

[0066] Any of the foregoing methods, alone or in combination, can beemployed in order to insure the stability of transgene expression intransgenic plants of the invention.

[0067] Kits. Also contemplated by the invention is a kit or kitscontaining insulator constructs in which the insulator elements of theinvention are provided in a DNA receivable vector or plasmid thatcontains or can be readily adapted by the user to contain theappropriate DNA elements for proper expression of a gene or genes ofinterest. The insulator element-containing plasmids or vectors of thekit may contain insulator elements, enhancers, a transcription unit, andthe gene or genes of interest may be inserted between the insulators, asdesired. Alternatively, the constructs of the kit may contain some orall of the necessary genetic elements for proper gene expression, orcombinations of these, and the remaining genetic elements may beprovided and readily inserted by the user, preferably between theinsulator elements in the construct. The insulator element-containingplasmids or vectors may be provided in containers (e.g. sealable testtubes and the like) in the kit and are provided in the appropriatestorage buffer or medium for use and for stable, long-term storage. Themedium may contain stablizers and may require dilution by the user.Further, the constructs may be provided in a freeze-dried form and mayrequire reconstitution in the appropriate buffer or medium prior to use.

[0068] The details of one or more embodiments of the invention are setforth in the accompanying description above. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the invention, the preferred methods andmaterials are now described. Other features, objects, and advantages ofthe invention will be apparent from the description and from the claims.In the specification and the appended claims, the singular forms includeplural referents unless the context clearly dictates otherwise. Unlessdefined otherwise, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs. All patents and publications citedin this specification are incorporated by reference.

[0069] The following EXAMPLES are presented in order to more fullyillustrate the preferred embodiments of the invention. These examplesshould in no way be construed as limiting the scope of the invention, asdefined by the appended claims.

EXAMPLE 1

[0070] Materials and Methods

[0071] Constructs. Standard DNA manipulation (restriction digestion,plasmid isolation, cloning, etc.) was performed as described by SambrookJ et al., Molecular Cloning: A Laboratory Manual, Second edition (ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

[0072] pGL419: Similar to pGL450, the fragment containing two repeats ofthe perfect palindromic insulator sequence and the CaMV 35S minimumpromoter (−51 region) with the TMV RNA Ω leader sequence wasPCR-amplified using pSH9 (Holtorf S et al., Plant Mol Biol 29, 637-646(1995)) as the template with the following two primers:

[0073] 5′GAAGATCTAGAATATATATATATTCGATAGAATATATATATATTCGCAAGACCCTT CC3′(SEQ ID NO:1) (underlined are the insulator sequences) and

[0074] 5′GGAATTCCATGGATCCCGGGTGTAATTGTAAATAG3′ (SEQ ID NO:2). Thisfragment was then fused with the GUS-MAS terminator to form pGL416,which was subsequently cloned into pPZP211 (Hajdukiewicz P et al., PlantMol Biol 25, 989-994 (1994)) at the BamHI and Xbal sites, resulting inpGL419.

[0075] pGL429: The CaMV 35S minimum promoter (−51 region) with the TMVRNA Ω leader sequence was PCR-amplified using pSH9 (Holtorf S et al.,Plant Mol Biol 29, 637-646 (1995)) as the template with two primers(5′GAAGATCTGATATCAAGCTTCGCAAGACCC3′ (SEQ ID NO:3) and5′GGAATTCCATGGATCCCGGGTGTAATTGTAAATAG3′ (SEQ ID NO:2)). The Ω leadersequence can enhance gene expression at the posttranscriptional level(Gallie D R et al., Nucleic Acids Res 15, 3257-3273 (1987)). The PCRproduct, upon cut with BglII and EcoRI, was cloned into pLITMUS28 (NewEngland Biolab, MA, USA) to form pGL400. A GUS-MAS terminator frompSG506 (Gan S, Ph.D. Thesis; Molecular characterization and geneticmanipulation of plant senescence (University of Wisconsin-Madison,Madison, 1995)) was then cloned into pGL400 at the NcoI and Xbal sitesto form the 35S minimum promoter-GUS-MAS terminator (pGL407), which wassubsequently cloned into the binary vector pPZP211 at the BamHI and XbaIsites to form pGL429. The 35S-NPTII was part of the pPZP211 sequence(Hajdukiewicz P et al., Plant Mol Biol 25, 989-994 (1994)).

[0076] pGL443: A fragment containing the CaMV 35S minimum promoter (−51region; with the TMV RNA Ω leader sequence)-GUS-MAS terminator wascloned into pPZP211 (Hajdukiewicz P et al., Plant Mol Biol 25, 989-994(1994)) at the XbaI site, resulting in pGL443 (and pGL429a in which thefragment was inserted in an opposite orientation compared with that inpGL443).

[0077] pGL446: The fragment containing two copies of insulator-35minimum promoter-GUS-MAS terminator was released from pGL416 (cf. pGL419construct) by using SpeI and XbaI, and was subsequently cloned intopPZP2 11 at the XbaI site, forming two constructs, one was named pGL446.

[0078] pGL450: The fragment containing two repeats of the perfectpalindromic lac Operator (lacO) sequence (Brown M et al., Cell 49,603-612 (1987)) and the CaMV 35S minimum promoter (−51 region) with theTMV RNA Ω leader sequence was PCR-amplified using pSH9 (Holtorf S etal., Plant Mol Biol 29, 637-646 (1995)) as the template with thefollowing two primers:

[0079] 5′GAAGATCTATTGTGAGCGCTCACAATGATAATTGTGAGCGCTCACAATTCGCAAGACCCTTCC3′ (SEQ ID NO:4) (underlined are the lacO sequences) and

[0080] 5′GGAATTCCATGGATCCCGGGTGTAATTGTAAATAG3′ (SEQ ID NO:2). Thisfragment was then cloned into pGL429a at the BamHI site, resulting inpGL450.

[0081] pGL469: Like pGL419, the fragment containing one copy of theperfect palindromic insulator sequence and the CaMV 35S minimum promoter(−51 region) with the TMV RNA Ω leader sequence was PCR-amplified usingpSH9 as the template with the following two primers:

[0082] 5′GAAGATCTAGAATATATATATATTCACTAGTTCGCAAGACCCTTCC3′ (SEQ ID NO:5)(underlined is the insulator sequence) and

[0083] 5′GGAATTCCATGGATCCCGGGTGTAATTGTAAATAG3′ (SEQ ID NO:2)). Thisfragment was subsequently cloned into pGL429a at the BamHI site to formpGL469.

[0084] Agrobacterium transformation. The above constructs weretransferred into Agrobacterium tumefaciens strain ABI using thefreeze-thaw method of An G, In Methods in Enzymology: Recombinant DNA,Wu R & Grossman L, eds., 292-305 (San Diego, Academic Press, 1987).Briefly, about 2-5 μg of each of the DNA constructs was added to a1.5-mL microcentrifuge tube containing 100 μL of competent ABI cells,mixed, and the mixture frozen in dry ice-ethanol bath, then placed in37° C. water bath for 5 minutes. 100 μL of YEP media was added into thetube, and the whole cells in the tube were plated on YEP platecontaining 100 mg/L spectinomycin (YEP: 10 g/L Bacto-peptone, 10 g/LBacto-yeast extracts, 5 g NaC 1; for plates, add 10 g/L phytoagar).

[0085] Plant transformation and cultivation. The Agrobacterium ABI cellscontaining the various constructs were used to transform Arabidopsis(ecotype Columbia) via vacuum infiltration as described by Bechtold N etal., C R Acad Sci Paris 316, 1194-1199 (1993). Briefly, about 200 mL YEPmedium with 100 μg/L spectinomycin was inoculated with a 10 mLpreculture of Agrobacterium harboring the respective construct. Thecells of overnight culture (28° C., 250 RPM shaker) with ˜1.4 OD₆₀₀ waspelleted by centrifugation and resuspended in 200 mL of infiltrationmedium (½×Murashige-Skoog salts, 1×B5 vitamins, 5% sucrose, 0.5 g MES,0.044 μM benzylaminopurine and 0.02% Silwet L-77, pH 5.7). Theresuspension was transferred into a 250-mL beaker, and a pot ofArabidopsis plants that had several flowers was invertedly submerged inthe cell suspension and vacuumed for 10- 15 min using an air vacuumpump. The plants were allowed to complete their life cycle and the seedswere harvested. After surface-sterilized with 70% ethanol containing0.1-0.2% Triton X-100, the seeds were sown on MS plates containingkanamycin (50 mg/L) and incubated 23° C. in an Arabidopsis growthchamber with 65% relative humidity under ˜150 μmol m⁻²s⁻¹ continuouslight from a mixture of cool white fluorescent (60%) and incandescent(40%) bulbs. The transgenic seedlings were either assayed for GUSexpression or transplanted into soil. The plants were grown in a plantgrowth facility under similar conditions.

[0086] GUS enzyme assays. The GUS assays in transgenic plants wereperformed histochemically and quantitatively according to the standardprotocol of Jefferson R A, Assaying chimeric genes in plants: the GUSgene fusion system, Plant Mol Biol Rep 5, 387-405 (1987). X-glucuronide(X-Gluc) was used as substrate for histochemical staining, and4-methylumbelliferyl-β-D-glucuronide for quantitative assays.

[0087] Nuclear protein extraction. Arabidopsis nuclear proteins wereisolated in a cold room using a protocol modified from Slomiany B A,BioTechniques 28, 938-942 (2000). Briefly, 1-2 g of 2-week oldArabidopsis seedlings were ground into powder in liquid nitrogen andhomogenized in 6 mL of extraction buffer (10 mM HEPES (pH 7.9), 10 mMKCl, 750 μM spermidine, 150 μM spermine, 0.1 mM EDTA, 0.1 mM EGTA, 1 mMDTT, 20 μM PMSF, 50 μg/mL antipain, 0.5 μg/mL leupeptin, 1 μg/mLpepstatin, 50 μg/mL chymostatin, 1 μg/mL aprotinin). After filteringthrough 44 μm nylon mesh, each 1 mL of filtrate was mixed with 1 μL NP40 and kept on ice for 10 minutes. Nuclei was pelleted (12,000×g, 3 min)and resuspended in 4×volume of nuclear storage buffer (50% glycerol, 20mM HEPES (pH7.9), 60 mM KCl, 0.5 mM EDTA). An equal volume of 0.8 Mammonium sulfate was then added to the suspension. After left on ice for20 min, the suspension was centrifuged (16, 000×g, 5 min), and thesupernatant was infiltrated through a G25 column (fine or medium) thathad been equilibrated with the column buffer (20% glycerol, 20 mM HEPES(pH7.9), 60 mM KCl, 0.5 mM EDTA) and aliquoted and stored in a −80° C.freezer.

[0088] Electrophoretic mobility shift assay (EMSA). DNA probes werelabeled with ³²P by using either Klenow enzyme fill-in or T4Polynucleotide kinase (Promega, Madison, Wis.) method. About 6,000-7,000CPM labeled DNA probes were incubated with or without 9 μg (unlessotherwise indicated) of nuclear proteins in a buffer (25 mM TrisCl(pH7.75), 10% glycerol, 0.2 μg BSA/μL, 0.1 mM EDTA, 0.05% NP−40, 1 mMDTT, 150 mM NaCl, and 0.2 μg Poly (dI:dC)/μL) on ice for 40 min. Thebinding reactions were run on 4% acrylamide gel. The gels were analyzedusing autoradiography and a phosphoimage analyzer (Fuji Model 2000).

[0089] DNA probes. The following oligonucleotides were synthesized andused in electrophoretic mobility shift assays (EMSA). The capitalletters indicate the palindromic sequences while the underlined aremutated nucleotides. Naturally mutated form NIm exists in theArabidopsis genome. The small letters are natural sequences flanking the16-bp palindromic insulator sequence in Arabidopsis. The nucleotides initalics are sequences filled by using the Klenow fill-in method. TABLE 1DNA probes used Probe # Name Sequence 1 L18 5′ATTGTGAGCGCTCACAAT3′ (SEQID NO:6) (Lac operator, 18 bp) 3′TAACACTCGCGAGTGTTA5′ 2 L315′cttctaATTGTGAGCGCTCACAATgaaaaag3′ (SEQ ID NO:7) (Lac operator, 31 bp)3′gaagatTAACACTCGCGAGTGTTActttttc5′ (SEQ ID NO:8) 3 NI16 (Nativeinsulator, 5′GAATATATATATATTC3′ (SEQ ID NO:9) 16 bp)3′CTTATATATATATAAG5′ (SEQ ID NO:10) 4 NI29 (Native insulator,5′cttctaGAATATATATATATTCgaaaaag3′ (SEQ ID NO:11) 29 bp)3′gaagatCTTATATATATATAAGctttttc5′ (SEQ ID NO:12) 5 NIm (Naturallymutated 5′cttctaGAATATATGTATATTCgaaaaag3′ (SEQ ID NO:13) insulator, 29bp) 3′gaagatCTTATATACATATAAGctttttc5′ (SEQ ID NO:14) 6 M1 (Mutatedinsulator, 5′cttctaACCTATATATATATTCgaaaaag3′ (SEQ ID NO:15) 29 bp)3′gaagat TGGATATATATATAAGctttttc5′ (SEQ ID NO:16) 7 M2 (Mutatedinsulator, 5′cttctaGAAGCGATATATATTCgaaaaag3′ (SEQ ID NO:17) 29 bp)3′gaagatCTTCGCTATATATAAGctttttc5′ (SEQ ID NO:18) 8 M3 (Mutatedinsulator, 5′cttctaGAATATCGCGATATTCgaaaaag3′ (SEQ ID NO:19) 29 bp)3′gaagatCTTATAGCGCTATAAGctttttc5′ (SEQ ID NO:20) 9 M4 (Mutatedinsulator, 5′cttctaGAATATCTATATATTCgaaaaag3′ (SEQ ID NO:21) 29 bp)3′gaagatCTTATAGATATATAAGctttttc5′ (SEQ ID NO:22) 10 M5 (Mutatedinsulator, 5′cttctaGAATATAGATATATTCgaaaaag3′ (SEQ ID NO:23) 29 bp)3′gaagatCTTATATCTATATAAGctttttc5′ (SEQ ID NO:24) 11 M6 (Mutatedinsulator, 5′cttctaGAATATATCTATATTCgaaaaag3′ (SEQ ID NO:25) 29 bp)3′gaagatCTTATATAGATATAAGctttttc5′ (SEQ ID NO:26) 12 M7 (Mutatedinsulator, 5′cttctaGAATATATAGATATTCgaaaaag3′ (SEQ ID NO:27) 29 bp)3′gaagatCTTATATATCTATAAGctttttc5′ (SEQ ID NO:28)

[0090] The palindromic sequence portion of the insulator sequence inTABLE 1 are as follows: NI 5′GAATATATATATATTC3′ (SEQ ID NO:9), NIm5′GAATATATGTATATTC3′ (SEQ ID NO:29), M1: 5′ACCTATATATATATTC3′ (SEQ IDNO:30), M2 5′GAAGCGATATATATTC3′ (SEQ ID NO:31), M3 5′GAATATCGCGATATTC3′(SEQ ID NO:32), M4 5′GAATATCTATATATTC3′ (SEQ ID NO:33), M55′GAATATAGATATATTC3′ (SEQ ID NO:34), M6 5′GAATATATCTATATTC3′ (SEQ IDNO:35), and M7 5′GAATATATAGATATTC3′ (SEQ ID NO:36)

EXAMPLE 2

[0091] Two copies of the 16-bp Palindromic Sequence have GenetucInsulator Activity that Blocks the 35S cis Elements from Directing theExpression of the P_(35Smini)-GUS

[0092] An eukaryotic promoter can be bidirectionalized by fusing aminimum promoter-gene construct at its 5′ end in opposite orientation.One example is pGL429, where the CaMV 35 minimum promoter-GUS-MASterminator chimeric gene (hereafter P_(35Smini)-GUS) is fused, inopposite orientation, with the 35S promoter directing the NPT II(kanamycin resistant) gene (construct 1 in FIG. 1). In addition, wegenerated 78 transgenic Arabidopsis plants harboring this pGL429construct and found that all of them displayed constitutive expressionof the reporter gene GUS. (TABLE 2 and FIG. 2A). This suggests that thecis elements of the 35S promoter exert their effect on the neighboringP_(35Smini)-GUS construct.

[0093] However, when two copies of a 16-bp palindromic sequence5′GAATATATATATATTC3′ (SEQ ID NO:9) were inserted between the 35Spromoter and the P_(35Smini)-GUS as shown in pGL419 (construct 3 in FIG.1), the effect of the cis elements of the 35S promoter on theP_(35Smini)-GUS was completely blocked because none of the 133independent pGL419 transgenic Arabidopsis plants showed the expressionof the reporter gene (TABLE 2 and FIG. 2C). TABLE 2 Expression of thereporter gene GUS in transgenic Arabidopsis lines Lines with GUSstaining Constructs Total lines (%) Comments pGL429 78  78 (100%)Bidirectional promoter pGL450 196 196 (100%) No insulating pGL419 133None (0%) Insulating pGL469 127 None (0%) insulating pGL443 186  60(32%) Enhancer trap, no insulating pGL446 194 None (0%) Insulating

EXAMPLE 3

[0094] TWO COPIES OF THE PALINDROMIC lac OPERATOR SEQUENCE FAILED TOBLOCK THE 35S PROMOTER FROM DIRECTING THE P_(35Smini)-GUS EXPRESSION

[0095] The data in EXAMPLE 2 suggest that the 2 copies of thepalindromic sequence block the 35S cis elements from directing theP_(35Dmini)-GUS. However, the blockage could result from (a) the spacereffect of the 2 copies of the palindromic sequence and/or (b) theformation of potential cruciform (cross-shaped) structure of thepalindromic sequence. To test these possibilities, we constructed pGL450in which 2 copies of the modified lac Operator (lacO) sequence were usedto replace the 2 copies of the 16-bp insulator sequence. The modifiedlacO sequence is 18 bp in length (2 bp longer than the insulator). Likethe insulator sequence, the modified lacO (5′ATTGTGAGCGCTCACAAT3′ (SEQID NO:6)) is also a perfect palindromic sequence (Brown M et al., lacrepressor can regulate expression from a hybrid SV40 early promotercontaining a lac operator in animal cells, Cell 49, 603-612 (1987)). Wegenerated 196 independent transgenic Arabidopsis lines harboring pGL450and found that all of the plants, like those plants harboring pGL429 butin contrast to those pGL419 plants, displayed constitutive GUSexpression (TABLE 2 and FIG. 2B). This data shows that the blockage ofthe two copies of the insulator is not due to its spacer effect, andthat the palindromic nature of the sequence is not necessary orsufficient for preventing the 35S cis elements from directing the GUSexpression.

EXAMPLE 4

[0096] One copy of the 16-bp Palindromic Sequence is Sufficient forGenetic Insulation

[0097] We further constructed pGL469 (construct 4 in FIG. 1) in whichonly one copy of the insulator sequence was used but otherwise theconstruct is the same as pGL419. All 127 independent transgenicArabidopsis lines generated showed no GUS expression (TABLE 2 and FIG.2D), indicating that one copy of the insulator is sufficient for playingits insulating role.

EXAMPLE 5

[0098] The Insulator Prevents Transgene from Position Effect ofNeighboring Genes of Plants

[0099] A position effect on a transgene is generally exerted by thepromoters/enhancers of neighboring genes. This position effect can bedemonstrated in either of the following two ways: one is to use a fullpromoter such as the constitutive 35S promoter fused to the reportergene GUS, transgenic plants containing this construct should display aspectrum of the GUS expression level, with some plants expressed at avery higher level and some other plants at a much lesser level. Theaddition of the insulator at the 5′ end of the promoter should result ina relatively even level of GUS expression with no or little variationamong all transgenic plants. This is a quantitative way to show positioneffect and the function of an insulator. However, this quantitativemethod can be complicated by the number of T-DNA insertion in the plantgenome and the number of T-DNA repeats in a single insertion site.Because of this complexity, we used an alternative way to demonstratethe position effect: the enhancer trap strategy.

[0100] In this enhancer trap strategy, a minimum promoter is fused to areporter gene such as GUS. This chimeric GUS gene is oriented towardsthe right border of T-DNA. When the construct inserts in the proximityof a chromosomal gene in plants, the neighboring gene promoter orenhancer of the plant genes may or may not direct the expression of thereporter, depending on the position of the insertion, i.e., in apopulation of enhancer transgenic lines, some plants will display noexpression of the reporter gene while some other transgenic lines mayshow the reporter gene expression in certain tissues at certaindevelopmental stages. We constructed pGL443 (an enhancer trap construct,see, construct 5 in FIG. 1) and pGL446 to test qualitatively if theinsulator can indeed eliminate the reporter gene expression resultingfrom position effect. pGL446 is identical with pGL443 except that thereare 2 copies of the insulator sequence at the 5′ end of the enhancertrap construct (construct 5 in FIG. 1). About 32% or 60 of the 186pGL443 transgenic Arabidopsis lines showed GUS staining at 2-week oldseedling stage (TABLE 2, and FIG. 2E). The GUS staining pattern (as wellas intensity) varied, with some lines showing staining in young leavesand roots while some other lines exclusively in roots or meristem(inserts in FIG. 2E). In contrast, there was no single line harboringpGL446 displaying any GUS staining (TABLE 2 and FIG. 2F).

EXAMPLE 6

[0101] The Insulator Interacts Specifically With Nuclear Proteins toForm a Complex

[0102] As described above, both the 16-bp insulator and the 18-bp lacOare perfect palindromic sequences that may potentially form cruciformsecondary structures, and the formation of the secondary structureitself may block the effect of other promoter elements on the reportergene expression. However, the lacO sequence failed to insulate thereporter gene expression (construct 2, TABLE 2 and FIG. 2B), suggestingthat the potential cruciform structure per se is not necessary orsufficient for insulating the effect of neighboring genes, and that theinsulating effect appears to be sequence specific.

[0103] We therefore hypothesized that the insulating role of theinsulator is achieved by interacting specifically with plant nuclearproteins. To test this hypothesis, we isolated Arabidopsis nuclearproteins and performed electrophoretic mobility shift assays (EMSA). Wefound that the ³²P-labeled insulator interacted with 5 μg of theArabidopsis nuclear protein extract to form a unique complex band on theacrylamide gels (Lane 2 in FIG. 3). The intensity of the complex wasincreased with 9 μg of the nuclear protein extract (Lane 3 in FIG. 3).The intensity of the complex was sharply decreased in the presence of0-, 2-, 20-, or 200-fold molar excess of the cold insulator competitor(Lanes 3 through 6). In contrast, the labeled lacO sequence did not formany complex (Lane 7 in FIG. 3).

EXAMPLE 7

[0104] Naturally Occurring A_(→)G Transition Singnificantly Reduced theFormation of the Insulator-Nuclear Protein Complex

[0105] The DNA region containing the insulator sequence in Arabidopsisare apparently recently duplicated (Gan S, Ph.D. Thesis; Molecularcharacterization and genetic manipulation ofplant senescence (Universityof Wisconsin-Madison, Madison, 1995)). The sequences of both copies areidentical except for 1-bp transition change in the palindrome region. Asshown in FIG. 4A, the A_(→)G transition makes the perfect palindrome (wenamed it native insulator, or NI) a non-perfect one (called NIm fornative insulator with a mutation). We were interested in knowing if thisnaturally occurring NIm sequence could also specifically interact withthe nuclear protein extract. The migration of the labeled NIm did shifton the acrylamide gel but the amount of the complex formed was reducedto 8.3% compared with that of non-mutant NI29 (Lane 4 vs. Lane 2 inFIGS. 4B and 4D).

EXAMPLE 8

[0106] The Effect of Articficial Mutations in the Insulator on theComplex Formation

[0107] We used the electrophoretic mobility shift assay (EMSA) tofurther investigate the function of nucleotides of the insulator ininteracting with the nuclear protein extract. We first made threemutated insulators, two of them with mutations in the potential “stem”region of the palindrome (M1 and M2 in FIG. 4A) and the other one inpotential “tetranucleotide loop” (M3). All these are transversionmutations. As shown in FIG. 4B and 4D, the ability of M1 interactingwith the nuclear extract was reduced to 50.5% of the natural insulatorNI29 while M2 remained near 95% of its binding activity. However, thetransversion mutations in the potential loop region (M3) completelyabolished the formation of the complex (Lane 10 in FIGS. 4B and 4D).

[0108] As shown in FIG. 4A, the M3 mutant that lost its ability ofinteracting with the nuclear extract has 4 transversional changes in thepotential loop region. To investigate the role of individualnucleotides, we further made 4 mutants, each mutant with only onetransversion (M4-M7 in FIG. 4A). Interestingly, except for the M5 thathad a reduced nuclear protein-binding activity (55.2%), other threemutants remained (M6) the same as or even outperformed over the nativeinsulator NI29. The M7 showed a 45% increase in nuclear protein-bindingactivity and the activity of the M4 was almost doubled (FIGS. 4C and4E).

[0109] Based upon these results, an initial consensus sequence wasobtained. TABLE 3 Sequence comparison Seq. Name SEQ ID NO Sequence NI(SEQ ID NO:9) 5′G A A T A T  A T A T ATATTC3′ NIm (Inactive) (SEQ IDNO:29) 5′G A A T A T  A T G T ATATTC3′ M1: (SEQ ID NO:30) 5′A C C T A T A T A T ATATTC3′ M2 (SEQ ID NO:31) 5′G A A G C G  A T A T ATATTC3′ M3(Inactive) (SEQ ID NO:32) 5′G A A T A T  C G C G ATATTC3′ M4 (SEQ IDNO:33) 5′G A A T A T  C T A T ATATTC3′ M5 (SEQ ID NO:34) 5′G A A T A T A G A T ATATTC3′ M6 (SEQ ID NO:35) 5′G A A T A T  A T C T ATATTC3′ M7(SEQ ID NO:36) 5′G A A T A T  A T A G ATATTC3′ Initial (SEQ ID NO:37)5′N₁ N₁ N₁ N₁ N₁ N₁ N₂ N₂ N₃ N₂ ATATTC3′ Consensus

[0110] The foregoing description has been presented only for thepurposes of illustration and is not intended to limit the invention tothe precise form disclosed, but by the claims appended hereto.

1 37 1 58 DNA Arabidopsis thaliana 1 gaagatctag aatatatata tattcgatagaatatatata tattcgcaag acccttcc 58 2 35 DNA Arabidopsis thaliana 2ggaattccat ggatcccggg tgtaattgta aatag 35 3 30 DNA Arabidopsis thaliana3 gaagatctga tatcaagctt cgcaagaccc 30 4 63 DNA Arabidopsis thaliana 4gaagatctat tgtgagcgct cacaatgata attgtgagcg ctcacaattc gcaagaccct 60 tcc63 5 46 DNA Arabidopsis thaliana 5 gaagatctag aatatatata tattcactagttcgcaagac ccttcc 46 6 18 DNA Escherichia coli 6 attgtgagcg ctcacaat 187 31 DNA Escherichia coli 7 cttctaattg tgagcgctca caatgaaaaa g 31 8 31DNA Escherichia coli 8 ctttttcatt gtgagcgctc acaattagaa g 31 9 16 DNAArabidopsis thaliana 9 gaatatatat atattc 16 10 16 DNA Arabidopsisthaliana 10 gaatatatat atattc 16 11 29 DNA Arabidopsis thaliana 11cttctagaat atatatatat tcgaaaaag 29 12 29 DNA Arabidopsis thaliana 12ctttttcgaa tatatatata ttctagaag 29 13 29 DNA Arabidopsis thaliana 13cttctagaat atatgtatat tcgaaaaag 29 14 29 DNA Arabidopsis thaliana 14ctttttcgaa tatacatata ttctagaag 29 15 29 DNA Arabidopsis thaliana 15cttctaacct atatatatat tcgaaaaag 29 16 29 DNA Arabidopsis thaliana 16ctttttcgaa tatatatata ggttagaag 29 17 29 DNA Arabidopsis thaliana 17cttctagaag cgatatatat tcgaaaaag 29 18 29 DNA Arabidopsis thaliana 18ctttttcgaa tatatatcgc ttctagaag 29 19 29 DNA Arabidopsis thaliana 19cttctagaat atcgcgatat tcgaaaaag 29 20 29 DNA Arabidopsis thaliana 20ctttttcgaa tatcgcgata ttctagaag 29 21 29 DNA Arabidopsis thaliana 21cttctagaat atctatatat tcgaaaaag 29 22 29 DNA Arabidopsis thaliana 22ctttttcgaa tatatagata ttctagaag 29 23 29 DNA Arabidopsis thaliana 23cttctagaat atagatatat tcgaaaaag 29 24 29 DNA Arabidopsis thaliana 24ctttttcgaa tatatctata ttctagaag 29 25 29 DNA Arabidopsis thaliana 25cttctagaat atatctatat tcgaaaaag 29 26 29 DNA Arabidopsis thaliana 26ctttttcgaa tatagatata ttctagaag 29 27 29 DNA Arabidopsis thaliana 27cttctagaat atatagatat tcgaaaaag 29 28 31 DNA Arabidopsis thaliana 28ctttttcgaa tatctatata tattctagaa g 31 29 16 DNA Arabidopsis thaliana 29gaatatatgt atattc 16 30 16 DNA Arabidopsis thaliana 30 acctatatat atattc16 31 16 DNA Arabidopsis thaliana 31 gaagcgatat atattc 16 32 16 DNAArabidopsis thaliana 32 gaatatcgcg atattc 16 33 16 DNA Arabidopsisthaliana 33 gaatatctat atattc 16 34 16 DNA Arabidopsis thaliana 34gaatatagat atattc 16 35 16 DNA Arabidopsis thaliana 35 gaatatatct atattc16 36 16 DNA Arabidopsis thaliana 36 gaatatatag atattc 16 37 16 DNAArabidopsis thaliana misc_feature (1)..(6) N can be any nucleotide 37nnnnnnnnnn atattc 16

What is claimed is:
 1. An isolated polynucleotide, comprising: (a) atleast one copy of a polynucleotide having the sequence set forth in SEQID NO:9; or (b) a polynucleotide which is a variant or fragment of thepolynucleotide set forth in SEQ ID NO:9, wherein the variant or fragmenthas a plant genetic insulator activity.
 2. The isolated polynucleotideof claim 1, wherein the polynucleotide comprises at least one copy of apolynucleotide having a set forth in the group consisting of SEQ ID NOS:1, 5, 9, 10, 11, 12, 15, 16, 17, 18, 21, 22, 23, 24, 25, 26, 27, 28, 30,31, 33, 34, 35 or
 36. 3. The isolated polynucleotide of claim 1, furthercomprising a replicable vector; into which the polynucleotide isinserted.
 4. The isolated polynucleotide of claim 3, wherein the vectoris an expression vector.
 5. The isolated polynucleotide of claim 3,wherein the vector is a plant vector.
 6. The isolated polynucleotide ofclaim 3, further comprising a host cell, in which the vector issituated.
 7. The isolated polynucleotide of claim 3, wherein the hostcell is a plant cell.
 8. The isolated polynucleotide of claim 3, furthercomprising a transgenic plant, in which the vector is situated.
 9. Theisolated polynucleotide of claim 8, wherein the plant is Arabidopsis ortobacco.
 10. The isolated polynucleotide of claim 3, further comprisinga transgenic seed, in which the vector is situated.
 11. A recombinantpolynucleotide, comprising: (a) at least one copy of a polynucleotidehaving the sequence set forth in SEQ ID NO:9; or (b) a polynucleotidewhich is a variant or fragment of the polynucleotide set forth in SEQ IDNO:9, wherein said variant or fragment has a plant genetic insulatoractivity.
 12. A method for expressing a polypeptide in a plant cell,comprising the steps of: (a) providing a vector comprising: (i) at leastone copy of either (A) a polynucleotide having the sequence set forth inSEQ ID NO:9; or (B) a polynucleotide which is a variant or fragment ofthe polynucleotide set forth in SEQ ID NO:9, wherein the variant orfragment has a plant genetic insulator activity; and (ii) a structuralpolynucleotide coding for a polypeptide; (b) inserting the vector into aplant cell, wherein the genetic insulator polynucleotide is recombinedinto the genomic DNA of the plant; and (c) allowing the plant cell toexpress the polypeptide.
 13. The method according to claim 12, whereinthe genetic insulator polynucleotide is located immediately upstream ofthe polynucleotide encoding the polypeptide.
 14. The method according toclaim 13, wherein the plant is Arabidopsis or tobacco.
 15. A method ofmaking a recombinant plant cell having reduced variability of expressionof a transgenic polypeptide therein, said method comprising: (a)providing a plant cell capable of regeneration; (b) transfecting saidplant cell with a polynucleotide construct comprising (i) a geneticinsulator polypeptide, comprising: (A) at least one copy of apolynucleotide having the sequence set forth in SEQ ID NO:9; or (B) apolynucleotide which is a variant or fragment of the polynucleotide setforth in SEQ ID NO:9, wherein the variant or fragment has a plantgenetic insulator activity; (ii) a transcription initiation region; and(iii) a structural polynucleotide encoding a polynucleotide; wherein thegenetic insulator polypeptide, the transcription initiation region andthe structural polynucleotide are operatively associated; wherein thepolynucleotide expression has a reduced variability as compared with aplant cell transfected with a polynucleotide construct that does notcontain the genetic insulator polypeptide.
 16. The method of claim 15,wherein expression of the transgenic polypeptide occurs in more of aplurality of the plant cells as compared to a plurality of the plantcells transfected with a polynucleotide construct that does not containthe genetic insulator polypeptide.
 17. A method for insulating theexpression of a transgenic polypeptide from cis-acting regulatoryelements in the plant chromosome into which the polynucleotide codingfor the expressed polypeptide has integrated, comprising: transfecting aplant cell with a polynucleotide construct comprising (a) a geneticinsulator polypeptide, comprising: (i) at least one copy of apolynucleotide having the sequence set forth in SEQ ID NO:9; or (ii) apolynucleotide which is a variant or fragment of the polynucleotide setforth in SEQ ID NO:9, wherein the variant or fragment has a plantgenetic insulator activity; (b) a transcription initiation region; and(c) a structural polynucleotide encoding a polynucleotide; wherein thegenetic insulator polypeptide, the transcription initiation region andthe structural polynucleotide are operatively associated; wherein thetransfected polynucleotide construct integrates into a chromosome of theplant cell; and wherein the expression of the polypeptide from theintegrated polynucleotide is insulated from cis-acting regulatoryelements in the plant chromosome into which the polynucleotide codingfor the expressed polypeptide has integrated.